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Microbiology assignment takes students to the world of the "unknowns"

For 10 weeks, Dr. Sean Coleman and his students spent their microbiology lab time learning various techniques used to identify bacteria. The labs were nice and predictable, because in the lab, everything is labeled, and you pretty much know what to expect when your run your tests. But in April, all of that changed, when Coleman sent the students out into the field and into the world of the "unknowns."

The assignment sounded pretty straightforward: Choose one or more bodies of water, collect samples, see if any coliform bacteria are found in the water, and if so, identify them.

Coliforms are a group of bacteria that are widespread in the natural environment, and which are present in large numbers in the feces of warm blooded animals. While some coliforms are pathogens themselves, most are not. However, their presence can be used to indicate contamination, which means that other pathogenic organisms may be present.

But sometimes, something that sounds simple turns out to be much more involved, and this was exactly what Coleman anticipated when the gave the assignment.

To complete their tests, the students first had to isolate bacteria from the samples they had collected by attempting to grow colonies of the bacteria. "We have these plates, which are relatively new, called Petrifilm from 3M, and what's nice about them is that they are specific to coliforms," Coleman said. "Pretty much anything gram-negative will grow on them, but only coliform bacteria will form these little gas bubbles. So you have this layer on top with these pink colonies with little gas bubbles around them and you know that's a coliform bacteria." Coleman said when the students found any coliform bacteria on their films, they could then use different techniques they had learned in lab to identify the bacteria.

"E. coli is easy to identify," he said. "Just put it on Levine's agar and if it turns it a green color…." He pointed to the poster prepared by students Elizabeth Small and Alex Holland, where there was a photograph of an Escherichia coli colony. But Coleman said that after that initial identification, things get a bit tougher. "The objective was to teach them real-world application," Coleman said, "but also teach them about identification of unknowns."

It took about six weeks of intense work, but the students' analysis was finally completed, and Coleman sent out an email inviting people to come to the poster presentations where they would present their results. "Are the waterways of the natural state safe for swimming? Boating? Do fecal coliform bacteria lurk in our waterways?" the email asked.

Sabrina Goddard and April Young took samples from three locations on the Arkansas River - here in Clarksville, at Little Rock, and at Tischner. "Tischner is where the White River meets the Arkansas River," Goddard explained. "We wanted to test Tischner because - I don't know if you know Hannah and Tyler - they went here. They found that the White River had high coliform counts, and we wanted to see if this transferred to the Arkansas River where they met." (See NASA grant enables U of O students to study river pollution.)

Goodard and Young found that the coliform levels at all three sites exceeded the levels established by the EPA as safe for contact, and at the Clarksville site, they far exceeded the "safe" level. They theorized that the higher levels at Clarksville were related to the proximity of the other sites to dams on the river, and to the predominance of livestock in Johnson County.

They also tested the bacteria they found to see if any had developed resistance to antibiotic. The good news, they said, was that while the levels were high at the three sites, none of the cultures showed any significant resistance to commonly used antibiotics.

Tess Montgomery, Laken Littlefield, and Colbey Russo tested a popular swimming area near campus, King's Canyon. They took samples from three sites - upstream from the pool, at the pool, and below the pool.

Based on the coliform counts they found, Montgomery said that none of the sites would be suitable for swimming under EPA guidelines. "We had the most [coliform] bacteria downstream," Littlefield said. She said the pool itself, where people swim, contained the second highest count.

Their samples contained E. coli as well as two species of Pseudomonas, which are the bacteria most commonly associated with swimmer's ear. However, she said that because of time constraints they weren't able to identify the Pseudomonas to the species level, nor to determine if the E. coli they found were the pathogenic strains. "The tests can take from two to five days to complete," she said, "so we spent several afternoons in the lab."

When asked if the higher water level caused by the recent rains could account for the higher coliform levels, Montgomery said, "It's interesting. We've thought about it a lot today - talked about it." She said that it is possible that as the water levels go down, one might see an increase in Pseudomonas, because those bacteria prefer warmer water temperatures. "But, at the same time, the contamination could have been caused by the rain, so we're not real sure," she said. "It would require more testing."

Bianca Cea and Kimberly Clabough took their samples at Horsehead Lake, where a U.S. Forest Service designated swim area is found. They sampled the swim area itself, as well as two other areas frequented by swimmers and fishermen - the waterfall and an area downstream from the waterfall.

Clabough said they began by using a staining process to determine the shape of the bacteria, and to indicate whether they were gram-negative or gram-positive. She said they followed those tests with more specific tests in an attempt to determine the species they had isolated from their samples. In addition to E. coli, they found a phytobacterium, which is a plant pathogen. However, it was somewhat frightening that Cea and Clabough found the E. coli they isolated exhibited some resistance to antibiotics.

"I don't like swimming in lakes in the first place, so after I did this, I definitely don't swim in lakes anymore!" Cea laughed. "Based on our results, it's better to go swimming in the fishing area or in the waterfall area than the swimming area."

Clabough said it's possible the recent rainfall may have impacted the bacterial counts in the lake. Although they did not make exact counts, their tests seemed to indicate that the counts would have been very high. "The lake was extrememly high," Clabough said. "There were geese there, so there are definitely a lot more animal interactions."

Alexa Holland and Elizabeth Small took six samples from three different sites right on Spadra Creek here in Clarksville. Site 1 was located downstream from a local processing plant; site 2 was the point where the stream runs right below the plant; and site 3 was upstream from the plant.

The pair found E. coli at their first sample site, downstream from the processing plant, while they found two unknown bacteria in their other samples. Unknown 1, collected at site 3, was identified as one of two Pseudomonas species, while unknown 2, taken from site 2, was either a Citrobactor diversus or Klebsiella oxytoca.

"That's as narrowed down as we could get without doing further testing," Small said. "But that's really good to get it down to two species."

Small said that the bacteria they found were not a cause for great concern, because they don't normally cause serious health problems. None of them showed any resistance to the antibiotics normally used against them. "We did a thermal death time," Holland said, "and all of them died after five minutes. So as long as you cook fish correctly, you shouldn't have any problems."

Elodie Adams and Megan Minniear found that looks can be deceiving when it comes to water quality. They sampled two small streams in the area - streams that appeared to be very pristine - and found large numbers of bacteria in both. "The first one looked safe and very natural, but it was actually very contaminated," Adams said.

Their tests showed that one of the streams contained E. coli, and they found two other unknown bacteria. Through a series of tests, they determined that one of the unknown bacteria was from the same family as E. coli, the Enterobacteriaceae. This family of bacteria includes many familiar pathogens. "If we had had the supplies and more time, we could have kept going," Minniear said.

Adams said that in the second stream, they found that their unknown bacteria were another pathogenic type that was typically associated with diarrhea, Enterobacter aerogenes. However, the pair found that in both cases the bacteria they isolated were not resistant to commonly used antibiotics.

The final poster was prepared by Tristan Cooper and Andrea Muffuletto, showing the results of their sampling along Big Piney Creek. They sampled at Long Pool Recreation area, at a walk-in station downstream from the forest, and at the point where the creek runs into Piney Bay.

They found that the count increased drastically as they moved downstream, going from 3 cfu/ml at Long Pool, to 31 cfu/ml by Piney Bay. However, they said the bacterial counts they found at their upstream sites were much lower than those being found by others in the class at their sites, and were probably not a cause for concern.

Cooper and Muffuletto also identified a bacteria from the Proteus genus as well as Morganella morganii which is known to cause a disease called Summer Diarrhea.

Coleman said he was very impressed with the students' work. "They wrote a proposal describing what they were going to do, and then they carried it out," he said. Because the assignment was so successful, Coleman said he plans to write it up as a method that may be used to teach the identification of unknowns and submit it for publication. And based on their test results, he said he plans to expand the assignment for the next class. "I know a few more tests that I want to have them do next time!" he said.